3.1-
Introduction:
Supramolecular hydrogels are formed by the self-assembly of hydrogelators1. These
supramolecular hydrogels contain a network of nanofibres and water (Fig. 3.1)2.
Hydrogels were defined by Flory in 1974 as “a coherent colloidal system of at least two
components, exhibiting mechanical properties characteristic of a solid, where both the dispersed component and the dispersion medium extend themselves continuously throughout the system”3. The amount of water in hydrogels is often more than 97%4.
Nowadays, supramolecular hydrogels have been used in many applications such as drug delivery5, 6, tissue engineering5-7, cell culturing8 and energy transfer9. They have also
been used in chemical sensing agents10. Currently, dipeptides with suitable functional
groups have a wide interest for use as hydrogelators. For example, dipeptides conjugated to aromatic group such as naphthalene or Fmoc have been used to form hydrogels11.
Figure 3.1: Self-assembly of hydrogelators to form hydrogel. Hydrogel preparation:
Supramolecular hydrogels are an emerging class of soft materials which are usually formed by the self-assembly of some small organic molecules. Supramolecular dipeptide hydrogels can be prepared by several different approaches. The main method used to prepare hydrogels is to dissolve the hydrogelators into an aqueous solution and then change the temperature12, pH13, or add an enzyme to start molecular self-assembly in
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water, resulting in hydrogelation. For example, Zhao et al. have shown hydrogelation by chemical or enzymatic conversion14, 15 such as phosphorylation of tyrosine1. In this
Chapter, we discuss the hydrogelation of a range of dipeptides.
3.2-
Experimental Section:
3.2.1-
Gel formation (Hydrogels based on dipeptide derivatives):
Hydrogels based on naphthalene, phenanthrol, anthraquinone, pyrene and carbazole dipeptide derivatives were prepared using different methods, solvent method (DMSO: water) or the pH switch method (GdL method)16, which are described below.
1- pH switch method (GdL method):
10 mg of dipeptide derivative were suspended in deionized water (2 mL) and an equimolar amount of NaOH (0.1 M) was added to the solution to dissolve the dipeptide. The solution was stirred for about 30 minutes or until a clear solution was formed. The pH of the solution was measured to be about 10 to 12. Measured quantities of glucono-
-lactone (GdL) were added to the solutions to control the pH to form gels. The samples were left to stand overnight to form hydrogels before measurement.
2- Solvent switch (DMSO: water):
The dipeptide derivatives (10 mg) were dissolved in 100 µL DMSO and then deionized water (1.9 mL) was added to give a final concentration of dipeptide of 5 mg/mL. The samples were left to stand overnight.
We have used a variation on the DMSO: water method where we used water of different pH to study their effect on the hydrogelation. Previous research illustrated that hydrogels using this method cannot be formed at high pH17-20, while other research
reported that hydrogels can be formed at high pH using a different method in the presence of salts21.
Dipeptide derivatives (10 mg) were dissolved in 100 µL DMSO and then made up to 2 mL with acetate buffer solutions of pH 3, 4, 5, 6 (1.9 mL), which were prepared as shown below (Table 1), were added to give a final concentration of dipeptide of 5 mg/mL. The samples were left overnight to form gels.
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Buffer solution preparation:
Acetate buffer solutions of pH 3, 4, 5 and 6 were prepared by mixing 0.1 M acetic acid with 0.1 M sodium acetate in different ratios22 as shown in Table 3.1.
pH Vol. of 0.1 M acetic acid Vol. of 0.1 M sodium acetate 3 982.3 mL 17.7 mL 4 847.0 mL 153.0 mL 5 357.0 mL 643.0 mL 6 52.2 mL 947.8 mL
Table 3.1: The proportions of 0.1M of acetic acid and 0.1 M of sodium acetate that were used to prepare acetate buffer solutions of pH 3, 4, 5 and 6 22.
3.2.2-
pH and pK
ameasurements:
An FC200pH probe (HANNA instruments) with a 6 mm x 10 mm conical tip was used to measure pH of the dipeptide derivatives. We utilised the GdL method to prepare the samples, whilst constantly measuring the pH (at a concentration of 5mg/ mL of dipeptide). We recorded the changes in the pH every 60 s overnight at room temperature.
To determine the pKa, water (5 mL) was added to the dipeptide derivatives (25 mg), and
then an equimolar amount of NaOH (0.1 M) was added to the solutions. The pKa was
determined using titration by adding a 0.1 M HCl (10 – 40 µL) every 5 minutes. The solution was stirred during the titration to prevent the formation of hydrogels23.
3.2.3-
Rheology studies:
Rheology is a method that was developed to characterise materials that possess both classical liquid-like and solid-like properties24. Rheology can measure and link the
properties of deformation of the solid state and flow of the liquid state. Rheology measures Gʹ and Gʹʹ. Gʹ refers to the storage modulus, which is a material’s ability to store energy, giving it solid-like properties. Gʹʹ refers to the loss modulus, which demonstrates the material’s ability to dissipate energy, giving it liquid-like properties. G’ is an order of magnitude larger than Gʹʹ when a material is considered to be a rigid hydrogel25.
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The rheology was studied for dipeptides that formed hydrogels using different methods for the preparation of hydrogels. 2 mL of the peptide solution as described above to prepare hydrogel was placed in a sample tube. The samples left to stand overnight to form hydrogel. After forming hydrogels, the rheology was studied using an Anton Paar Physica MCR101 rheometer. All tests were performed at room temperature. Frequency sweeps at constant strain (0.5 %) were measured between 1 rad s-1 and 100 rad s-1
using a cup and vane geometry. The measurements of Gʹ and Gʹʹ with gelation were carried out at constant frequency (10 rad/s) at 25οC. Rheological data was acquired for
each of the successful hydrogelators and compared.
3.2.4-
Fourier-transform Infra-Red Spectroscopy (FT-IR):
FT-IR spectroscopy is a technique that studies the absorption of infra-red light. It measures the absorbed light by the sample to produce peaks at specific wavelength. The absorption peaks correspond to the frequencies of vibration between specific bonds in the material. This result identifies important functional groups that should be present in the product such as carbonyl groups, carboxylic acids and amide bonds26. Infra-red
spectra of hydrogels were collected using a TENSOR series Bruker FT-IR spectrometer, at 2cm-1 resolution averaging 64 scans. Samples were prepared in D2O, adding 1.9 mL of
D2O to dipeptide dissolved in 100 µL DMSO. Samples were left for a minimum of twelve
hours to form hydrogels. The hydrogels were transferred onto the IR plates without damaging the supramolecular structure. We also prepared samples using the GdL method in D2O, H2O in both the wet and dry gel states as well as the as-synthesised
dipeptide. The FT-IR was carried out by Lin Chen, University of Liverpool.
3.2.5-
Scanning electron microscopy (SEM):
SEM images were recorded using a Hitachi S-4800 FE-SEM at 3 KeV. Diced silicon wafers (Agar) were used to deposit a portion of the hydrogels, prepared as described previously (the hydrogel of compound 40 was prepared in GdL method). The hydrogel was allowed to stand on the silicon wafer while it was air-dried for 1 hour. The silicon wafers were placed on aluminium stubs with an Agar adhesive tape. A 5 nm gold layer was used deposited with a sputter coating machine for 1 minute at 5 mA current. To avoid charging, a low voltage SEM was used (0.5 to 1 keV) at a 1.5 to 3 mm working
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distance with the deceleration mode. The SEM was carried out by Andre Zamith Cardoso, University of Liverpool.
3.3-
Result and discussion:
3.3.1-
Hydrogel and Rheology:
3.3.1.1- Hydrogelation of naphthalene dipeptides:
The ability of naphthalene dipeptides to form hydrogels was studied previously using different methods2, 23, 27, 28. Here, we have used a solvent switch method (DMSO: water)
and the GdL method because they have been used previously as effective methods to form hydrogels with combinations of different aromatic groups and amino acids, naphthalene and Fmoc dipeptides 23, 29. Here, we attempted to use these methods with
dipeptides containing different aromatic groups and different hydrophobicity to study the formation of hydrogels and compared the data with that previous collected. The first method used was a solvent method (DMSO: water). In this method, 100 µL of DMSO was added to 10 mg of dipeptide derivative. Then, 1.9 mL H2O was added to the solution. We
have also used this method with buffered water (pH 3, 4, 5 or 6) in order to study the formation of hydrogels at different pH. The other method we used to form the gels is the GdL (glucono--lactone) method16. Here, the self-catalysed hydrolysis of GdL in water
lowers the pH of the solution slowly30. The hydrolysis mechanism opens the lactone
ring, producing gluconic acid which donates protons to the dipeptides, allowing non- covalent interactions to dominate and self-assembly to occur.
We are interested in the different methods of forming gels as these can be used to change the properties of the final material11. In the solvent switch method, the self-
assembly starts quickly after adding the water to the solution of peptide in DMSO and the hydrogel can form in a few minutes. This method might be useful for drug delivery application because it can be used to form an injectable hydrogel.31 On the other hand,
in the GdL method, the formation of a hydrogel occurs after few hours. This method is useful to study and understand the self-assembly process and the mechanical properties of the hydrogel.19 The solvent switch method at different pH was used for compounds 10 – 25 (Fig. 3.2). Some dipeptides formed hydrogels and others did not using the solvent switch method with different acetate buffers. The results of these methods are
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shown in Table 3.2, which shows the rheological data, i.e. storage modulus (Gʹ) and loss modulus (Gʹʹ) for the samples that formed a gel. According to the rheology, some of dipeptide derivatives have high Gʹat low pH such as compound 12, 13, 16 and 17. This means these dipeptides are good hydrogelators. Others, which have low Gʹ, are poor hydrogelators. These results agreed with previous research17-20. For instance, Chen et al.
reported that dipeptides conjugated to aromatic groups can form hydrogels at low pH, whereas at high pH no gels are formed. Moreover, some peptides did not form hydrogel at all pH such as compound 10 and 11.
There are other factors that are known to affect hydrogel formation such as hydrophobicity and pKa17, 19, 20. Here, it can be clearly seen that an increase in
hydrophobicity and pKa can lead to the formation of hydrogels17-20. For example, 16 and 17 formed gels at all pH (pH 3, 4, 5, 6). In contrast, 15 formed hydrogel at pH 3 and pH 4, which agrees with other research that reported hydrogels formed at low pH using the DMSO method11. Similarly, two naphthalene dipeptide derivatives based on 2-naphthol
formed gels. One of them formed hydrogels at all pH, compound 13, whereas compound 12 was only able to form hydrogels at pH 3 and pH 4. These results show that the hydrophobicity cannot be used as a simple guide to whether a gel is formed, as we can see that compound 15 which contains a bromine group and is more hydrophobic than compound 13.
123 | P a g e Sample name pH 3 pH 4 pH 5 pH 6 Gʹ (Pa) Gʹʹ (Pa) Gʹ (Pa) Gʹʹ (Pa) Gʹ (Pa) Gʹʹ (Pa) Gʹ (Pa) Gʹʹ (Pa) Compound (11) - - - - Compound (10) - - - - Compound (12) 22100 5100 14200 3000 - - - - Compound (13) 4000 200 2000 100 3000 300 3100 400 Compound (14) - - - 20000 4000 Compound (15) 6200 600 6000 500 - - - - Compound (16) 12000 3000 24000 7300 20000 4000 6000 600 Compound (17) 21000 00 2000 3000 14000 2000 15000 3000
Table 3.2: The Gʹ and Gʹʹ using pH switch method at pH 3, 4, 5, 6 for 2-napthol and 6-bromo-naphthol derivatives. (–) refers to dipeptides that did not form gels. G’ and G’’ were measured using frequency sweep at
10 rad/s.
Table 3.3 shows the results of the formation of the hydrogels using the solvent switch and GdL methods. It can be clearly seen that eleven dipeptides out of nineteen formed hydrogels using GdL method, while nine dipeptides formed hydrogels using the solvent switch method. In the GdL method, the pH was measured (before adding GdL) to be between 10 and 12 for the solution of dipeptide derivatives. After adding GdL and forming hydrogels, the pH was measured to be between 3 and 4. The pH drop allows the formation of gels19. Other research reported that hydrogels can be formed at high pH
using a different method (adding calcium salts)21.
Table 3.3 also shows that some dipeptides formed transparent gels and others formed turbid gels. The factors that cause the turbidity could be the molecular structure of the dipeptides. For example, dipeptides that formed turbid hydrogels have high molecular structure, while dipeptides that formed transparent gels have low molecular structure (Fig. 3.3). This may due to the aggregation of the peptides that have high molecular structure into larger structures, which lead to increase in light scattering, leading to form a turbid hydrogel. Also, the turbidity can be affected by the concentration of the dipeptide. We noted that when we decrease the concentration of the hydrogelators, the turbidity decreased, presumably due to a decrease in the aggregation of the fibres.
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Figure 3.3: Examples of hydrogel images that formed a turbid and transparent gel. Compound 17 (Left) formed a turbid gel and compound 12 (Right) formed a transparent using the GdL method. It shows the effect
of the molecular structure on the turbidity.
Furthermore, it can be seen that any change in the peptide structure such as substitution position or order of amino acid sequence can affect and change the ability of the molecules to form a hydrogel. For instance, if we compare between dipeptides that have similar amino acids and different numbers or different substitution positions of a bromine atom, we note that they have different gelation results. For example, compound 14, 18 and 22, where compound 14 (bromine at position 6 on the naphthalene ring) did not form hydrogel, while compound 18 (where there are bromines at position 1 and 6) and 22 (the bromine at position 1) formed a turbid hydrogels using the solvent switch method. As a result, it can be clearly seen that a small change in the peptide structure can affect and change the formation of the hydrogel. Also, as described above, we noted that the hydrophobicity did not correlate to hydrogel formation. For instance, compound 12 formed a turbid hydrogel, whereas compound 20 and 25 which are more hydrophobic than 12 did not form hydrogel using the solvent switch method. These results agreed with other examples that discussed in the previous research29. Furthermore, Chen’s group19 have prepared similar naphthalene dipeptide
hydrogel with different amino acid sequence using the GdL method. For example, their results showed that 2-napFVOH and 6Br-napFVOH formed turbid hydrogels, which are Similar to our results that showed compounds 13 and 17 which they have similar structure and different amino acid sequence formed also turbid hydrogels. Also, precipitate was formed from compound 11, while a crystal was formed from Chen’s compound which has different amino acid sequence (2-napAV)19.
Compound 30
Compound 33
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It is clear that all the dipeptides that form gels with the solvent switch method also form gels with the GdL method. However, dipeptides 15, 25 and 30 all only form gels with the GdL method. It is still not clear why some peptides formed gel using only the GdL method. However, we expect that the method of preparing the hydrogel can modify the properties of the hydrogel formed11.
Dipeptide Gel formation DMSO: H2O
Gel formation GdL Molecular weight
LogP Compound 10 - Precipitate - Precipitate 400.36 1.390 Compound 11 - Precipitate - Precipitate 372.31 0.619 Compound 12 √ Turbid Gel √ Transparent Gel 358.46 0.832 Compound 13 √ Turbid Gel √ Turbid Gel 448.45 2.080 Compound 14 - Precipitate - Transparent Gel 479.36 2.140 Compound 15 - Precipitate √ Turbid Gel 451.31 1.360 Compound 16 √ Transparent Gel √ Transparent Gel 437.46 1.570 Compound 17 √ Turbid Gel √ Turbid Gel 527.45 2.820 Compound 18 √ Turbid Gel √ Turbid Gel 558.20 2.920 Compound 19 √ Turbid Gel √ Turbid Gel 530.21 2.141 Compound 20 - Precipitate like gel √ Turbid Gel 516.18 2.354 Compound 21 √ Turbid Gel √ Turbid Gel 606.30 3.602 Compound 22 √ Turbid Gel √ Turbid Gel 479.36 1.520 Compound 23 √ Turbid Gel √ Turbid Gel 451.31 1.400 Compound 24 √ Transparent Gel √ Transparent Gel 437.46 1.620 Compound 25 - Precipitate √ Transparent Gel 527.45 2.870 Compound 30 - Precipitate √ Turbid Gel 384.47 1.490 Compound 31 - Precipitate - Precipitate 356.44 0.717 Compound 32 √ Transparent Gel - Gel precipitate 342.39 0.932 Compound 33 √ Turbid Gel √ Transparent Gel 432.51 2.178
Table 3.3: Dipeptides that formed gels and did not form gels using GdL method and DMSO: water method. (–) means did not form gels, (√) means formed gels. This is a qualitative observation.
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We have also studied the rheology of the gels that are formed using frequency sweeps and the strain sweeps to demonstrate the mechanical properties of the formation of the hydrogel at different conditions. Figure 3.4 (a, b) shows example data for 16. The hydrogels were prepared using the GdL method at concentration of 5 mg/mL of the dipeptide. Figure 3.4 (a) shows the strain sweep. The data shows that the gel still has solid-like properties until a strain of about 5% and the deformation of the gel starts after this point. Here, we can see a fast drop of G’ and this indicates that after this point, the hydrogel has more liquid-like properties than solid-like properties, indicating breakdown of the structure of the hydrogel. A crossover point at a strain of about 5% is typical for this kind of hydrogel23. Figure 3 (b) shows the frequency sweep of the same
hydrogel. From Figure 3.4 (b), it can be seen that the hydrogel has strong solid-like properties, with a G’ of over 104 Pa. G’’ was approximately 103 Pa. As expected for this
kind of hydrogel18, 23, 27, 32, both G’ and G’’ were relatively independent of frequency. This
data shows that compound 16 is a good hydrogelator. Furthermore, we noted that the G’ is different in the strain and frequency sweep, although the data for the same hydrogelator. These differences might be simply because we have prepared two different samples of the same hydrogelator, one to measure the strain sweep and the other to measure the frequency sweep. This may lead to errors in the preparation.
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Figure 3.4: An example of studying the mechanical properties of compound 16. (a) strain sweep (b) Frequency sweep.
(a)
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Table 3.4 shows the rheology studies for the dipeptides that formed hydrogels (Table 3.4 reports the Gʹ and Gʹʹ values). Gʹ and Gʹʹ for compound 20 were low, although a gel was still formed.
In some cases, the rheology was very different for the gels prepared by the different methods. For example, compound 17 formed a turbid gel with both methods, but has higher G’ when the gel is formed by the GdL method than by the solvent switch method (27 kPa and 14 kPa respectively). However, the Gʹof the gel formed from 12 was 26 kPa using the GdL method and 25 kPa using the solvent switch method, showing that the GdL method does not always result in stronger gels. Figure 3.5 shows examples of some dipeptides that formed gels using the GdL method and the solvent switch method where transparent gels are formed. Transparency will be a key parameter for uses in energy transfer (see Chapter 4).
Peptides GdL DMSO
Gʹ (Pa) Gʹʹ (Pa) Gʹ (Pa) Gʹʹ (Pa)
Compound 12 26000 6000 25000 6000 Compound 13 6000 300 6000 1000 Compound 16 23000 4000 16000 3000 Compound 17 27000 3000 14000 2000 Compound 18 4000 400 5000 4000 Compound 19 6000 800 1400 200 Compound 20 2000 300 20 15 Compound 21 3000 400 92000 11000 Compound 22 3000 200 8000 400 Compound 23 300 40 1000 130 Compound 24 7000 1000 12000 2000 Compound 25 15000 3000 - - Compound 32 3000 300 1000 200 Compound 33 60000 7000 5000 500